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Blood, 1 May 2001, Vol. 97, No. 9, pp. 2917-2917
CORRESPONDENCE
To the editor:
Induction of proliferation and augmented cytotoxicity of  
T lymphocytes by bisphosphonate clodronate
We read with interest the article by Kunzmann et al, which
states cytoreductive effects of aminobisphosphonates mediated by gamma
delta T cells (  cells).1 All tested
aminobisphosphonates induced significant expansion of cells.
This proliferative response was interleukin-2 (IL-2) dependent, whereas
activation of T cells (up-regulation of CD25 and CD69) occured
in the absence of exogenous cytokines. Pamidronate-treated bone marrow (BM) cultures of patients with multiple myeloma showed significant reduced plasma cell survival compared with untreated cultures. When
T cells were depleted in BM before treatment, cytoreductive effects were completely abrogated. These results are supported by
findings that we gained from pure T cell
cultures.2 We found that T cells isolated from
peripheral blood can kill human neuroblastoma cells efficiently
(100% specific lysis at an effector-to-target cell [E/T] ratio of
20:1). The cytotoxic effects of T cells were not restricted to
neuroblastoma cells. cells were also cytotoxic against human
Burkitt lymphomas Daudi (32.7% specific lysis at an E/T ratio
of 20:1; identical to the data from Kunzman et al1) and
Raji (10.3%), human colon carcinoma cell line Colo 205 (23.1%),
erytholeukemia cell line K562 (100%), and human neuroepithelioma cell
line SK-N-MC (100%).2 These data raise the possibility of
using this in a clinical setting. We enriched TCR -expressing T
cells out of peripheral blood mononuclear cells (PBMNCs) by positive
selection. T cells were magnetically and fluorescence labeled
using a hapten-modified anti-TCR antibody and
fluorescein isothiocyanate-conjugated antihapten microbeads (both from
Miltenyi Biotech, Bergisch Gladbach, Germany). Subsequent positive
selection was carried out with LS+/VS+ columns
according to the protocol supplied by Miltenyi Biotech. The efficiency
of positive selection was evaluated by flow cytometric analysis. Fifty
milliliters of peripheral blood from a healthy adult donor normally
yielded approximately 2 to 14 × 106 T cells. The
cells were then tested in stimulation assays to determine
optimum propagation and optimum proliferation for ex vivo generation in
order to get reasonable amounts for clinical applications. The
proliferation-inducing capacities of clodronate (Disodium(dichloromethylene)-diphosphonatetetrahydrate, a
bisphosphonate like Isopentenylpyrophosphate, but nontoxic), IL-2, PHA,
IL-15, OKT3 (antiCD3; bound), or combinations of these were tested in pure T cell cultures. Our choice to use clodronate as a
proliferation-inducing substance was also based on the observation made
by Berenson et al,3 who had reported that survival rates
were significantly raised in a group of patients who had recieved
bisphosphonate pamidronate as infusions additionally to salvage
chemotherapy. We were interested in whether these findings were in
context with T cell cytotoxicity and based on a possible
selective (specific) induction of T cell proliferation. We chose clodronate for our study instead of pamidronate since
it is a classic bisphosphonate, and substantial differences in the
hematologic response to initial treatments with aminobisphosphonates have been reported.4-6 The optimized concentration of
clodronate for in vitro assays was predetermined using different
concentrations (75, 60, 45, 30, and 15 µg/mL) in proliferation
assays. Best results were obtained with a single dose of 37.5 µg of
clodronate per milliliter of growth medium. Higher concentrations of
clodronate resulted in a strong inhibitory effect on cell
proliferation.2 (Optimized doses are obtainable in vivo:
recommended medication in vivo is 300 mg clodronate
intravenously within 2 hours. This equals an initial concentration of
75 µg clodronate per milliliter of blood in a patient with 4 liters
of blood.) 6-3[H]-thymidine incorporation was measured on day 14 of
the culture period. Clodronate, PHA, or OKT3 alone showed effects that
were comparable to untreated control cells (proliferation 1.7, 1.6, and
1 times control, respectively). IL-15 and IL-2 increased proliferation
7 and 20 times, respectively. Maximum proliferation was induced by the
combination of IL-2/clodronate or IL-2/OKT3 (increase of proliferation
by a factor of 35.8 and 57.8 times that of control, respectively). Both
combinations clearly exhibited more than just an additive effect. When
IL-2-treated T cells were set as control, increases in
proliferation by additional treatment with clodronate, PHA, and OKT3
were 74.4%, 38.5%, and 182%, respectively. A comparison between
proliferative effects on T cells of the most effective dual
combination (OKT3 and IL-2) with triple combination (OKT3, IL-2, and
clodronate) after 14 days in culture revealed a 15% enhancement of
proliferation with the latter (in 3 individual assays) (Figure
1). This might be due to an additional,
different stimulation mechanism induced by clodronate.
Isopentenylpyrophosphate or BCG (BCG Vaccine Behring; Chiron-Behring,
Marburg, Germany) failed to increase proliferation of OKT3 and
IL-2-treated cells.
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| Figure 1.
Proliferation of T lymphocytes.
Fifty thousand T cells were seeded per well in 96-well
microtiter plates. Growth medium was supplemented with indicated drugs
and proliferation measured by 6-3[H]-thymidine
incorporation assay. Replicates were set up 10-fold. Columns represent
the number of counts per minute (mean ± SD) of
6-3[H]-thymidine incorporation.
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Since we had tested clodronate for induction of proliferation, it was
an interesting proposition to determine whether clodronate modifies the
cytotoxic activities of T cells, in addition to its
proliferative properties. T cells were grown with and
without clodronate in the presence of IL-2. Cell cytotoxicity
was determined at E/T ratios of 20:1 to 1.2:1 in BATDA release (a
nonradioactive cell cytotoxicity-determining assay with a high
sensitivity comparable to chromium release).7 No
difference in cytolytic activity compared with an IL-2-only-treated
control group was seen after 5 days, but clear effects were detectable
on days 13, 21, and 30 (Figure 2).
Clodronate-treated T cells clearly exhibited higher cytotoxic
activities toward neuroblastoma cells than untreated control cells (at
an E/T ratio of 20:1, killing was 31.3% ± 9.9% SD). This
was also observed at the lowest tested E/T ratio of 1.2:1, where
cytotoxicity was even 48.6% ± 17% SD higher than cytotoxicity of
the control group. Our data agree with Kunzmann et al1 that
proliferation response of T cells to (amino)bisphosphonates are
IL-2-dependent. Kunzmann et al1 found only a slight effect on T cell proliferation by bisphosphonate clodronate, compared with the dramatic increase of cells in PBMNC cultures induced by
aminobisphosphonates. This seems reasonable since PBMNC cultures offer
the possibility of cross-reactions with cytokine producing/releasing immune competent cells and/or interactions with a variety of other effector cells and T cells. Thus, effects mediated by
aminobisphosphonates might be primary or the end of a cascade of
reactions in PBMNC cultures. Secondary side effects cannot be excluded.
But nevertheless, in a T cell-restricted system,
bisphosphonates induce measurable effects on proliferation and
cytotoxicity of T cells. These data and the capacity of
efficient killing of human malignant cells by T cells suggest an
important role for them in immunological strategies of cancer
therapy, where aminobisphosphonates and bisphosphonate clodronate
mediate T-cell proliferation and activation.
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| Figure 2.
Cytotoxicity of T cells against neuroblastoma
cells.
T lymphocytes were cultured for a period of 30 days with IL-2 or
with IL-2 and clodronate. Columns represent the percentage of specific
lysis in a cytotoxicity-determining assay (BATDA release)4
compared with control (maximum release) at different time points.
Results are shown for E/T ratio 20:1.
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Karin Schilbach and Andreas Geiselhart
University Childrens Hospital Department of
Hematology and Oncology Tuebingen, Germany
Rupert Handgretinger
St Jude Children's Research Hospital Division of
Stem Cell Transplantation Memphis, TN
References
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J Immunother.
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3.
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Effect of first treatment with aminobisphosphonates pamidronate and ibandronate on circulating lymphocyte subpopulations.
J Bone Min Res.
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Blomberg K, Hautala R, Lovgren J, Mukkala VM, Lingqvist C, Akerman K.
Time-resolved fluorometric assay for natural killer activity using target cells labelled with a fluorescence enhancing ligand.
J Immunol Meth.
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